Production of penicillin



PRODUCTION OF PENICILLIN Donald John Darlington Hockenhull and George Desmond Wilkin, Uxbridge, and Anthony Richard John Quilter, South Chingford, London, England, assignors to Glaxo Laboratories Limited, Greenford, England, a British company No Drawing. Application March 6, 1953, Serial No. 340,915

Claims priority, application Great Britain March 10, 1952 1-1 Claims. (Cl. 195-36) the antibiotics which come Within the general term penicillin are those known as penicillin G, penicillin X, and

penicillin F, of which penicillin G is the most important.

The penicillins may be represented by the general formula where R can represent various groups; in the case of penicillin G, R is a benzyl group.

Penicillin is produced by the fermentation of a penicillin producing mould in a suitable nutrient medium, normally under aerobic deep culture conditions and very extensive research has been carried out to discover suitable nutrient media which give rise to maximum yields of penicillin. The media at present used for the commercial production of penicillin are based on corn steep liquor and contain besides this material, water, nutrient salts, a carbohydrate such as lactose and if desired other organic material such as maize oil. The composition of media based on corn steep liquor required to support the growth of a penicillin producing mould are well known and such a medium is herein referred to as a corn steep liquor medium.

It is also well-known that the yields of penicillin obtained by fermenting *a penicillin-producing mould in a suitable corn steep liquor medium can be markedly increased by including in such media certain organic substances such as phenylacetic acid, phenylacetamide, fl-phenylethylamine (see British Patent No. 586,930) and such substances have become known as penicillin precursors. Such substances are believed to supply the fragment R in the above mentioned formula for penicillin since the nature of the precursors efiects the nature of the penicillin obtained; thus if the medium contains phenylacetic acid improved yields of benzyl penicillin (penicillin G) are obtained, while if p-hydroxyphenylacetic acid is employed a greater proportion of p-hydroxybenzyl penicillin results. Other precursors of the general formula (where R is a member of the group of radicles consisting of hydrogen, methyl, ethyl, lower alkyl ethe-rsand acyloxy United States Patent 9 Patented Sept. 4, 1956 derivatives thereof) are referred to in specification No. 613,492for example phenylacetylethanolamine. A substance containing in its molecule the fragment R of the ,penicillins and which when included in a suitable corn steep liquor medium gives rise to increased yields of penicillin is-herein referred to as a penicillin precursor.

United States Patent No. 2,437,918 describes the incorporation of substances there referred to as sulphite containing compounds in penicillin fermentation media, and while referring in general terms to many sulphur containing compounds, deals in specific terms with metallic sulphites; the specification refers in one instance to the use of thiosulphates but no examples are given of the employment of thiosulphates in a corn steep liquor medium nor of the results to be obtained thereby; the addition of such sulphite containing compounds is sugg'ested more for so-called synthetic media than for corn steep liquor media, and indeed it is suggested that the presence of the sulphite enables corn steep liquor to be dispensed with. To the best of our knowledge the use of sulphites has not found favour with penicillin manufacturers in the United Kingdom or in the United States of America.

As a result of considerable experiment we have now made the discovery that for optimum penicillin production using a suitable corn steep liquid medium it is necessary to provide in the medium both a source of the fragment R of the penicillin molecule and a source of sulphur (presumably for the thiazolidene ring). Thus our observations show that the addition of a penicillin precursor by itself while giving rise to increased yields of penicillin, will give further increases of yield if a suitable source of sulphur is present. Similarly while the presence of a source of sulphur by itself in a medium which does not contain a penicillin precursor is helpful, the presence of a precursor still further increases the yield. These discoveries are important at high levels of added substance. Thus for example prior to the present invention optimal yields were obtained with additions of phenylacetylethanolamine of the order of 0.1% and increased quantities over this figure did not give much extra penicillin; we now find that by also including a source of sulphur We are able to use higher levels of penicillin precursors and obtain higher yields of penicillin than can be obtained using any quantity of precursor Without such addition; thus in effect by having both the source of sulphur and the penicillin precursor present together in the medium it appears that the mould is able to make more use of each of these two individual substances than would be the case if only one of them were present. The selection of a suitable source of sulphur for the purpose of the present process has required careful research and several sources such as those proposed in United States Patent 2,437,918 have been tested. We find however that thiosulphates alone appear to give satisfactory results and that other sulphur-containing compounds, such as cysteine, sulphates, sulphites and the like are not satisfactory. Our invention is further only of value under conditions of culture in which high yields are normally obtainable namely deep culture conditions. In brief it may be said that the discovery on which the present invention is based is that by fermenting under deep culture conditions a penicillin producing mould in a suitable cornsteep liquor medium containing both a thiosulphate and a penicillin precursor, the mould is more able to take advantage of the precursor and the sulphur in its .synthesis of penicillin with the result that higher levels of these two additives can be used with the resulting production of higher yields of penicillin than hashitherto been possible. Under optimum conditions yields of greater than 2,000 international units per may'be consistently obtained. It is to-be noted that our experiments show that the high yields obtainable by the process according to the invention cannot in general be achieved With synthetic media which contain a source of sulphur and a penicillin precursor but which contain no corn steep liquor.

According to the present invention therefore we provide a process for the production of penicillin inwhich a penicillin producing mould is fermented under deep culture conditions in a corn steep liquor medium containing a thiosulphate and a penicillin precursor as herein defined.

According to a further feature of the invention the penicillin precursor is phenylacetic acid, phenylacetamide, phenylacetylethanolamine, or p-phenylethylamine.

The thiosulphate employed should be at least partially soluble in water and its cationic portion should cause no poisoning of the mould nor destruction of the penicillin produced at the level at which it is employed. It will be easy to determine by preliminary shake-flask experiments whether any particular thiosulphate is suitable; we prefer to employ sodium thiosulphate, potassium thiosulphate, ammonium thiosulphate or magnesium thiosulphate.

The preferred penicillin producing moulds for the purpose of the present invention are Penicillium notatum and Penicillium chrysogenum.

As stated above we are able to use levels of penicillin precursor higher than those previously employed and we prefer to employ more than 0.1% and preferably 0.2% of the precursor, the upper limit being determined by the slight toxic efiect on the mould of certain precursors.

It is to be noted that some precursors are rapidly consurned during fermentation without necessarily being employed by the mould for penicillin synthesis and at present it is normal in such cases to add further quantities of precursor as the fermentation proceeds; such a precursor is for example phenylacetic acid.

The addition of further quantities of precursor from time to time during fermentation may also be necessary in the process according to the present invention although it is not necessary using the type of precursor referred stituent which a given strain of mould will tolerate can be increased by acclimatisation of the strain to the particular conditions.

For the better understanding of the invention the following experimental work is quoted for the purposes of illustration and without limiting the general statements hereinbefore contained:

EXAMPLE 1 Experiments comparing the efiect of sodium thiosulphate at 0.08% level with varying levels of phenylethylamine and phenylacetamide were carried out.

For these experiments the strain of Penicillium chrysogenum used was PM3.

The basal medium was as follows:

Lactose 3.5%. C. S. L. solids 3.5% (by weight). KH2PO4 0.4%. Chalk (Analar) 1.0%. Maize oil 0.16 ml. per flask added separately.

Sodium thiosulphate and the phenyl ethylamine or phenylacetamide as required were added by weight to portions of this bulk medium. 7

0.15% phenylacetamide-S days culture Experiment No. Titre Mean With thiosulphate0.08% and 0.15% phenylacetamide:

Experiment No. Titre Mean 3 gig B15 4 fig 480 0.3% phenylacetamideNo thiosulphate:

Experiment N0. Titre Mean With thiosulphate 0.08%:

Experiment No. Titre Mean egg 565 4 Phenylacetamide6 days culture [Level=0.15%-No thiosulphata] Experiment No. Titre Mean 9 fig 295 10 gg 316 With thiosulphate 0.08%:

Experiment No. Titre Mean .11 Y 485 435 12 gig 545 [Leve1=0.3%-No thiosulphate.]

Experiment No. Titre Mean With thiosulphate 0.08%:

Experiment No. Titre Mean Phenylacetamide7 days culture. With 0.08% thiosulphate:

[Level 015%.]

Experiment No. Titre Mean Experiment No. Titre Mean Phenylethylamine-6 days culture th'iOSlilPhiitB 0.08%: [Level 0.15%.]

Experiment No. Titre Mean Experiment N o. Titre Mean 7 I a; r 550 9 520 in g g I 430 I With 0.08% thiosulphate:

{Level 03%;] v i Experiment No. Titre Mean Experiment No. Titre Mean 550 210 a9 485 4520 21 lggg 285 40-. fig 415 e2 290 295 25 [Level 0.3%.]

(108% thlosulphate: Experiment No. Titre Mean 41 470- 470 ExperimentNo. Titre 'Mean 455 455 23 e00 600 24 555 With 0.08% thiosulphate:

Phenylethylamme gave results as follows: Expat ant No Tim Mean -5 days vulture-level 0.15% 560 V M 4a. 555

e D Experiment No. Titre Mean 40 640 605 315 p '[-Leve1 0.6-%.] 7' M n 0 Experiment No. Titre Mean p 45 j With 0.08% th10su1phate: V g8 450 460 r H 41o 435 Experiment No. Titre Mean 4,27 495 495 With 0.08% .thiosulphate: 28 465 465 r [Lev 81 03% Experiment No. Titre Mean ExperimentNo. Titre Mean 47 550 48 v 565 29 310 310 en e85 '385 l v Phenylefhylamine-7 days culture With 0.08% thiosulphate: {Level 015%.] e

Experiment-(No. Titre Mean Experiment-N0 Titre Mean 0 l 225 7 31 A 370 1420 49 5330'} 280 $2 50 460 460 32 49o 495 With thiosuiphate (0.08%): [Level 0.6%.]

H u .x r r. Experiment No. Titre 7 Mean Experiment No. Titre :Mgan

7 [Level 0.3%.} These results, .showing approximately 2001. U./ml. in-

crease in titre at Sand 6 days for the thiosulphate flasks, Experiment No. Titre Mean were statistically significant.

Since it was not known whether theeflz'cct was due to a 53 540 "540 peculiarity of the particular batch of cornsteep used, 475 fourteen other batches were tested in shake-flasks with Y and without the addition of sodium thiosulphate and in with thwslllphate every' case it was found that an increase in titre was 7 obtained by the addition. Experiment No. Titre Mean 10 Sodium thiosulphate was next tested in 5-litre fermenters. For these experiments a medium containing a 55 575 570 higher concentration of both lactose and cornsteep solids 222 was used: 56 i 510 545 5 Percent Lactose 3.5 wave! 05%] Cornsteep solids 3.5

Experiment No. Titre Mean 04 Phenylacetylethanolamine 0.1 51 I 660 660 58" g 450 Maize 011 0.25 I To this medium was added 0.08% of sodium thiosulphate, W1th'th1osulphate (0.08%): equivalent to, 0.02% of sulphur as in the shake-flask experiments. Experiment N0. Titre Mean After 96 and 108 hours, fermentation the following.

7 titres were attained: 575 50 V E 510 m 515. ,7 90 hours, 108 hours,;

3 7 Bottle 1. U. I. U. rh1.

Fermentations carried out in '5-litrefermenters- 17 1 430 1 620 The following microbiological titres were obtained from Q g f: 338 11% five-litre fermenters on the same basal medium as that "I 11550 11460 used for the shake flasks. Phenylacctamide was added. 35 3- 35g fig at 0.15 and 0.3%.

fi? iifififig'lg fif gf Control tests carried out in exactly the same manner L Home 40 but without the sodium thiosulphate glve the following 0.15% phen- 0.3%phen- 0.15% phen- 0.3% phenresults: ylacetamlde ylacetamlde ylacetamide ylacetamide 2 3 ggg 23g Bottle 92 hrs. 116 hrs 790 760 870 1, 030 900 880 1,030 1,150 25 1 200 750 020 740 1,150 1,010 26 1,140 850 030 720 1, 430 1, 070 090 930 780 810 980 000 28 840 1, 270 20: 910 1,100

EXAMPLE 2 gouolwing f i h employed for experimmts Further experiments-in S-Iitrefermenters were done in wlth P eny may 6 am we a'medium of the following composition:

Percent Lactose 3 r 7 Percent Cornsteep solids, V I 3 Lactose Chalk V 1 Corn steep liquor solids (by weight) 3.5 mp0,; Q4 Phenylacetylethanolamine .'..J..... 0.2 Phenylacetylethanolamine 0.1 KH?PO4 Maize 011 .(by .volume), 0.25 sodmmlthwsulphate. .60 Chalk 1.0 a number of shake-flask experiments were conducted Maize oil 025 using Penicillium chrysogenum var. brevisterigma (see n Umted. States Patent 5 3 5? half i F g In these experiments the precursor was increased from were y 5. f a g 2 ffi p 3 m 0.1% to,0.2% in order to sustain the increased penicillinmm 105 P ate 7 f e f were ,65 producing potential of the sodium thiosulphate addition. as 0 The following titres are indicativeof the yields achieved.

Control, -I-Thiosul- Incubation time 1 .I.U./ml. phate, r v

' LIL/ml. Bottle 96 hours, 8hours, a g g '70 Y I. U./m1. I. U./m1.

, 1,340 1,310 a i 7 V 1,32% gggg "5;-.- Y gggg 1, 10 a 1 112 1 212 11165 11310 V '0 The same medium was then found to be equally beneficial for other strains of P. chrysogenum. Using two difierent strains the following titres were obtained:

Experimental data were next obtained levels of corn steep liquor and lactose. titres were obtained:

using different The following Treatment 96 hours 108 hours 3.5% Lsetosenu 3.5% Lactose--- {4.0% CSL 4.0% Lactose {4 5% 4:0% Lactose Percent Lactose 3.5 Corn steep liquor solids (by weight) 3.5 Phenylacetylethanolamine 0.2 KH2PO4 0.4 Sodium thiosulphate 0.08 Chalk 1.0

EXAMPLE 3 A number of experiments were carried out using phenylacetic acid as precursor. The conditions and results were as follows:

Medium:

Lactose 3.5%. C. S. L. solids 3.5%. KHzPOe 0.4%. Chalk 1.0%. Maize oil 0.16 ml. per flask added separately. Sterilized 20 minutes at 15 lbs. per square inch pressure.

Strain: P. chrysogenum, P. M. 3. Precursor: Three levels of neutralized phenylacetic acid added aseptically at 24 hrs.

RESULTS 0.12% Phen- 0.24% Phen- 0.48% Phenylacetic Acid ylacetic Acid ylacetic Acid 5 With N With N 0 With No 0.08% Thio- 0.08% Thio- 0.08% Thio- Thios Thiosul- Thiosul' sulphate sulphate sulphate phate phate phate 10 days:

350 251 420 50 14 7 440 255 430 190 18 6 Flask 3 251 375 295 14 Mean 395 251 408 178 15 6 It will be noted that at the 0.24% level of precursor, its toxic efit'ect is removed on the addition of the thiosulphate.

We claim:

l. A process for the production of penicillin which comprises fermenting a penicillin-producing mould under deep culture conditions in a corn steep liquor-containing medium containing both a penicillin precursor and an at least partially water-soluble thiosulphate, the amounts of said precursor and said thiosulphate in said medium being sufficient to produce an increased yield of penicillin but insufiicient to cause substantial inhibition of the fermentation.

2. The process defined in claim 1 in which said medium contains at least 0.1% by weight of said precursor and at least 0.01% by weight of sulphur in the form of said thiosulphate.

3. The process defined in claim 1 in which said medium contains at least 0.2% by weight of said precursor and at least 0.02% by weight of sulphur in the form of said thiosulphate.

4. The process defined in claim 3 in which said thiosulphate is sodium thiosulphate.

5. The process defined in claim 3 in which said thiosulphate is potassium thiosulphate.

6. The process defined in claim 3 in which said thiosulphate is ammonium thiosulphate.

7. The process defined in claim 3 in which said thiosulphate is magnesium thiosulphate.

8. The process defined in claim 3 in which said penicillin precursor is phenylacetic acid.

9. The process defined in claim 3 in which said penicillin precursor is phenylacetamide.

10. The process defined in claim 3 in which said penicillin precursor is phenylacetylethanolamine.

11. The process defined in claim 3 in which said penicillin precursor is fi-phenylethylamine.

References Cited in the file of this patent UNITED STATES PATENTS McCormack Mar. 16, 1948 Behrens Apr. 27, 1948 OTHER REFERENCES 

1. A PROCESS FOR THE PRODUCTION OF PENICILLIN WHICH COMPRISES FERMENTING A PENICILLIN-PRODUCING MOULD UNDER DEEP CULTURE CONDITIONS IN A CORN STEEP LIQUOR-CONTAINING MEDIUM CONTAINING BOTH A PENICILLIN PRECURSOR AND AN AT LEAST PARTIALLY WATER-SOLUBLE THIOSULPHATE, THE AMOUNTS OF SAID PRECURSOR AND SAID THIOSULPHATE IN SAID MEDIUM BEING SUFFICIENT TO PRODUCE AN INCREASED YIELD OF PENICILLIN BUT INSUFFICIENT TO CAUSE SUBSTANTIAL INHIBITION OF THE FERMENTATION. 